*3.1.2 Attachment and growth*

When the adhesion is effective, the bacteria start to grow by taking advantage of the available nutrients. Following this stage, biological events dominate bacterial adhesion to the surface. This is the outcome of the expression of a number of genes that are in the position of producing surface proteins such as porins [62].

#### **Figure 1.**

*Schematic illustration of the key steps in biofilm formation: (a) planktonic bacteria attaching to the surface, (b) motility factor inhibition, (c) extracellular polymeric substances (EPS) generation, and (d) biofilm maturation and dispersal. The figure is adapted and modified from a previous study with copyright permissions [58].*

The polysaccharides used to create the EPS layer are transported with the help of porins. Since the biofilm matures, microbial cells start to connect to each other via the release of autoinducer signals (AIs) [63]. This communication is critical due to an established biofilm can comprise up to 100 billion bacterial cells per milliliter. The cells are divided into identical different groups, each of which is accountable for a specific task [64]. Another frequently observed phenomenon in a growing biofilm is the formation of high and wrinkled structures. However, this causes lateral pressure on the cells by pulling it towards one another. The dead cells in the biofilm concentrate in the areas that promote vertical bulging, which helps in releasing this pressure [65].

#### *3.1.3 Metabolism*

The metabolic process of the biofilm modifications with changes in the environment of the biofilm throughout the primary phase of growth of biofilm, when the metabolic activity is strong and subsequently declines as growth progresses [66]. The complex diffusion channels are employed as the cell population grows to transport nutrients, oxygen, and further components required for cell growth. These channels are used to transport the metabolic wastes and debris. In fact, shear stress has a significant impact on the expression of genes involved in glycolysis [67]. The bacteria that form biofilms have a propensity to ingest foreign DNA, which could eventually lead to the expression of exogenous proteins [68]. Furthermore, it has been demonstrated that several genes involved in the biosynthesis of fatty acids were downregulated as the biofilm formed [69]. These findings show that biofilm-forming cells have a very different metabolism from planktonic cells.

#### *3.1.4 Dispersion*

The final step is dispersion, which involves the destruction of the biofilm and the sessile cells, allowing them to resume their motile forms. Finally, biofilm makes use of its disruptive forces to spread throughout, and the bacteria colonize new regions and develop [70].

### **3.2 Biofilm regulation**

This protective biofilm structure is comprised of proteins and smaller molecules which are strung together to form larger, more robust polymer units of sugars (polysaccharides), macromolecules like DNA, and lipids [71]. This EPS helps the bacteria contained within the structure survive by supplying nutrients, removing waste products, and preventing harmful antimicrobial molecules, antibodies, and host inflammatory cells from getting or interacting with the bacteria. A developed biofilm additionally helps in the ongoing maturation and eventual spreading recolonization of the encased bacteria, prevents molecules too large to pass through the structure, and provides a diffusion barrier to small molecules like antibiotics (**Figure 2**) [4, 73–76].

Defensive EPS structure of bacterial biofilm protects bacterial hybridization, tolerance, and gene expression by subverting the natural infection inflammatory response in order to get rid of the body of bacteria and support the survival of bacteria [75]. It is noteworthy that this protective structure can repel treatments and promote continued biofilm growth, even if the biofilm is chemically or mechanically fractured into microcolonies, rendering the bacteria within the structure virtually invincible unless the structure is solubilized and removed. The protective EPS structure of the

*Formation, Regulation, and Eradication of Bacterial Biofilm in Human Infection DOI: http://dx.doi.org/10.5772/intechopen.114177*

#### **Figure 2.**

*The general mechanism of biofilm resistance to antimicrobials. (A) Biofilm matrix provides a diffusion barrier to small molecules like antibiotics. (B) Inactivation of antibiotics by enzymes of the biofilm matrix. (C) Persister cells in the deeper layer of biofilm inducing adaptive SOS reaction and hence developing further resistance. The figure is adapted and modified from a previous study with copyright permissions [72].*

biofilms protects bacterial hybridization, tolerance, and gene expression patterns and promotes bacterial survival by preventing the body's own inflammatory response that is designed to get rid of bacteria [18]. This procedure enables the biofilm to rapidly mature and develop impenetrable to conventional treatments as well as unculturable using conventional culture methods. The biofilm may function passively as a reservoir for pathogenic bacteria which are typically polymicrobial in nature, or it can take a more active role by encouraging an expanding area of inflammation and pathogenic tissue damage that favors the progression into overt infection as the biofilm develops into a more mature insoluble biomass, encouraging protected bacterial growth, mutation, and proliferation through sophisticated cell-to-cell and cell-to-surface interactions between the host and the biofilm [77–81]. Over time, a portion of the biofilm's bacteria disperses as fresh-roving bacteria and micro-bacterial aggregates that release and spread, acting as the foundation for new biofilm colonies [82].

#### **4. Bacterial biofilms: Pathogenicity and properties of the bacterial biofilms**

#### **4.1 Pathogenicity**

It is well-established that biofilms contribute to the virulence of pathogens. According to statistics from the Centres for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH), the prevalence of disease caused by biofilms is believed to be between 65% and 80%, particularly in developed nations

[83]. Several food-borne pathogens including *E. coli, Salmonella, Yersinia enterocolitica, Listeria,* and *Campylobacter* create biofilms on the surface of food or storage equipment. Furthermore, potentially pathogenic bacteria such as *Staphylococcus aureus, Enterococcus faecalis, Streptococcus, E. coli, Klebsiella,* and *Pseudomonas* thrive on catheters, prosthetic joints, mechanical heart valves, and so on. As a result of their periodic escape from the said focus these organisms may cause persistent diseases [83, 84]. The localized depletion of nutrition in a biofilm has been proposed as an inducer of cell release or detachment from the biofilm in *Pseudomonas aeruginosa* [85]*.* However, microbially produced gas bubbles, cross-linking cations, growth status, contact surface material, shear stress, quorum sensing, and lytic bacteriophage activation have all been identified as major contributors to biofilm detachment. They can be life-threatening causing endocarditis and infections in people with cystic fibrosis, in addition to infecting long-term indwelling devices like heart valves and joint prostheses [86].

Numerous bacterial toxicities in the human body such as the development of dental plaques, infections of the middle ear in children, urinary tract infections, gingivitis, and contact lens infections are caused by biofilms. The biofilm formation takes place on contact lenses and ultimately it leads to contamination [9, 87].

#### **4.2 Properties of the bacterial biofilms**

The bacterial biofilms cause pathogenicity through a variety of unique properties.

#### *4.2.1 Variation in phase*

Small colony variations (SCVs) are a colony phenotype highlighted by small size, slow development, and virulence gene downregulation and have been identified as a pathogenic mechanism for various bacterial species such as *S. epidermidis* and are often linked with chronic infections [88]. Biofilms have the unique potential to generate bacterial subpopulations that are shifted to a dormant state and are known as small colony variations (SCVs). They also have low catalase activity which interferes with oxidative metabolism. SCVs generate noticeable morphological changes in biofilms, increasing adhesion, auto-aggregation, hydrophobicity and pathogenicity. These variations contribute to biofilm survival in extreme environmental conditions [58]. SCVs appear to be less sensitive to antibiotics and the immune system possibly due to their ability to survive intracellularly and induce a more anti-inflammatory setting due to higher Inter Leuken-10 (IL-10) release [89].

#### *4.2.2 Efflux pumps*

Efflux pumps are present in the periplasmic space within the bacterial membranes and have a negative influence on antibiotic accumulation and their presence is critical in pathogenesis in biofilm [90]. The mutations in regulation proteins or promoters result in the production of these efflux pumps which causes pathogenicity. These efflux pumps are energy-dependent and on the basis of mechanism by which they derive energy are generally categorized into two groups. The primary efflux pumps get their energy from constant hydrolysis of ATP whereas the secondary efflux pumps get their energy from chemical gradients created by protons or ions like sodium ions [91]. The increased expression of these efflux pumps in biofilm has also been linked to pathogenesis in *P. aeruginosa* biofilms by causing antibiotic resistance [92].

*Formation, Regulation, and Eradication of Bacterial Biofilm in Human Infection DOI: http://dx.doi.org/10.5772/intechopen.114177*

#### *4.2.3 Alterations in membrane protein expression*

The outer membrane channel proteins existing in bacterial membranes play an important part in transferring hydrophilic particles from the outer atmosphere to the periplasmic space in biofilm of bacteria [93]. The presence of outer membrane proteins (Omps) allows for macromolecular interaction between the cell and the environment. These proteins are established in the outer membrane of bacteria in biofilm. A larger channel outer membrane protein such as OmpF can be used in place of OmpC because it has a smaller diameter. This change limits the entry of bigger compounds with high hydrophobicity such as carbenicillin. In contrast, small hydrophilic molecules such as imipenem can pass through the OmpC channels. In biofilm, differential expression of outer membrane protein-coding genes occurs which contributes to antibiotic resistance and pathogenicity [94].

## **5. Bacterial biofilm and its eradication**

Many types of nanoparticles with therapeutic effects against bacterial biofilm infections can be categorized according to their chemical composition or their healthcare purposes. The nano-formulations have a high attraction to the bacterial cells and the capacity to penetrate biological barriers like biofilm because of their small size, large surface area, and highly sensitive nature [95]. The sizes of the NPs are sufficiently small to penetrate into biofilms and microbial cell walls, while the large surface area of the NPs enables the effective loading of drugs [96]. Although the exact strategy of NPs reducing biofilm formation is not completely elucidated yet, multiple studies have described different processes by which NPs impact bacterial cells and biofilms (**Figure 3**). There are various common types of nanoparticles used for biofilm eradication, which are listed and discussed below.

#### **5.1 Metal-based nanocomposites**

Metal nanoparticles (MNPs) are often employed in antibacterial and antibiofilm studies due to their inherent nature, structure, and large surface-to-ratio, enabling control in fabrication, approach, and modification of their physical and chemical characteristics [98]. MNPs demonstrate significantly greater antibacterial activity compared to their micro-sized counterparts, although NPs, like common antimicrobials, lack the ability to recognize sensitive and resistant microorganisms [96]. However, their non-specificity is also one of their drawbacks because they can also attack commensal bacteria [99]. Metal oxide nanoparticles (MONPs), silver nanoparticles (AgNPs), gold nanoparticles (AuNPs), and various other metal-based nanocomposites (NCs) have shown their efficiency in preventing biofilm formation through a distinct inhibitory mechanism [96, 99].

#### *5.1.1 Metal oxide NPs*

Metal oxide nanoparticles (MONPs) that demonstrated antimicrobial activity consist of iron oxide (Fe3O4), zinc oxide (ZnO), titanium oxide (TiO2), silicon oxide (SiO2), selenium oxide (SeO2), and aluminum oxide (Al2O3). The majority of the NPs impacts on microbial cells involve cellular membrane breakdown resulting from NP-cell surface interaction and consequent leak of cell substance [100]. Metal oxide

#### **Figure 3.**

*The primary mechanisms of nanoparticles (NPs) in biofilm eradication. (A) Interaction with functional components of biofilm via the released ions. (B) Production of reactive oxygen species (ROS) that cause bacterial destruction and EPS breakdown. (C) Antimicrobial-loaded polymeric nanoparticles penetrate into the biofilm and deliver drugs to the bacterial cell. (D) the photothermal effect, which occurs in the presence of near-infrared (NIR) light irradiation, causing an increase in local heat, which acts efficiently alongside EPS and bacterial cells. (E) Liposomes encapsulate the antimicrobial and fuse with cell membranes, allowing the antibiotic to be released directly inside the bacterial cell. The figure is adapted and modified from a previous study with copyright permissions [97].*

NPs (MONPs) also mediate mechanisms for DNA and RNA destruction, the production of ROS, and the discharge of poisonous substantial metal ions [98]. The primary antibacterial activity of these NPs is related to oxidative stress, which is caused by the formation of ROS on the outer layers of metal oxides and subsequent breakdown of cell membranes, structure of cells, and molecules [98, 101]. The effects of oxidative stress can harm the proteins that contribute to attachment and biofilm development. Furthermore, it suppresses the development of genes that are important for bacterial cell attachment on surfaces and biofilm development [102].

#### *5.1.2 Silver nanoparticles (AgNPs)*

AgNPs are commonly used as antimicrobial agents and have greater antibacterial activity than some antibiotics as well as employed in clinically developed devices, tubes, and dressings for wounds [103]. In addition to exhibiting antimicrobial properties, AgNPs possess a large surface-to-mass ratio, which makes them an attractive choice for use as single layers on the surfaces of biomolecules [104]. The antibacterial effect of AgNPs is considered to be caused by the NPs' breakdown and the release of Ag+ ions, which attach to the cell membrane and depolarize the cell wall while changing the permeability and negative charge of the membrane. Additionally, when Ag+ ions penetrate the target bacterium, they cause the oxidation and breakdown of cellular components, the reduced activity of respiratory chain enzymes, and the formation of ROS, which hinders the recombination of DNA and the fabrication of ATP [98, 105].

*Formation, Regulation, and Eradication of Bacterial Biofilm in Human Infection DOI: http://dx.doi.org/10.5772/intechopen.114177*

#### *5.1.3 Gold nanoparticles (AuNPs)*

AuNPs are more efficient against biofilm compared to AgNPs because they have a lower hydrophobicity index, which reduces the growth of biofilm [106, 107]. The antimicrobial process of AuNPs is believed to involve affecting the membranes of bacterial cells and inhibiting ATPase production, which leads to metabolic degradation, as well as hindering the ribosome component attaching to tRNA, attacking nicotinamide, and impacting the bacterial respiratory chain [105]. In addition to having antimicrobial qualities, AuNPs also exhibit photothermal characteristics when exposed to nearinfrared (NIR) light. This is because accumulated AuNPs absorb light in a red-shifted manner, which causes a dramatic increase in localized heat. Consequently, this represents yet another potent means of eradicating bacteria from the infectious biofilm without damaging the tissues that surround it because their cells require a greater amount of heat due to their larger size than bacterial cells [108, 109].

#### *5.1.4 Metal-polymer nanocomposites (MNCs)*

The fabrication of MNPs as polymer nanocomposites increases their stability and efficiency. According to Nagvenkar et al. [110], adding ZnO NPs to polyvinyl alcohol (PVA) polymer enhanced the stability and efficiency of the ZnO-PVA nanofluid against *S. aureus and E. coli* [110]. The toxic effects of ZnO can be decreased through its incorporation into different materials. Banerjee et al. [111] showed that doping pancreatin (PK) on ZnONPs (ZnONPs-PK) reduced the toxic effects of ZnO and increased its antibacterial and anti-biofilm efficiency while decreasing its virulence towards MRSA [111]. Depan and Misra [112] found that incorporating titania NPs into silicone decreased *S. aureus* life and adhesive abilities by 93% when compared to stand-alone silicone [112]. Silicone-TiO2 NPs were also more effective at breaking down the biofilm; after 6 hours of incubation, the biofilm completely disintegrated. Wang et al. [113] observed that AuNPs incorporated with graphitic carbon nitride (g-C3N4) could improve H2O2 efficiency by causing peroxidase-like activity that effectively breaks down H2O2 to OH radicals, leading to demonstrated biofilm destruction and prohibited biofilm development in vitro, reducing the growth of *E. coli and S. aureus*, and potentially accelerating the healing process [113]. Recently, capping MNPs with polysaccharides obtained from other microbes like yeast and algae has been shown to be effective [114].

## **5.2 Polymer-based nanoparticles (PNPs)**

Polymer nanoparticle-based antimicrobial delivery systems are whatever they are commonly referred to as in regards to their functionality. Despite the fact that the chemical composition could be organic, inorganic, or even a mixture of both, their enhanced antibacterial transport is due to their improved stability, capacity for modification, formation at the site of infection, and monitored release ability, along with boosted cytocompatibility and biodegradable properties [98, 115, 116]. PNPs have a distinct advantage over MNPs because medications can be maintained within their cavity, allowing the drug to be delivered to the target region, whether confined or entrapped [99, 117]. Based on this, PNPs are available in two shapes, nanospheres and nanocapsules, with sizes ranging from 100 to 500 nm. The nanosphere is a polymeric matrix that contains the drug that has been adsorbed in it. The drug can be entrapped in small cavities or adsorbed onto the polymer wall of nanocapsules, which have an oily core and a polymeric shell around them [117].

### **5.3 Natural and synthetic polymer-based nanoparticles (PNPs)**

PNPs can be either synthetic or natural, like chitosan, polycaprolactone, polylactic acid, and polylactic-co-glycolic acid (PLGA). Chitosan, a cationic heteropolysaccharide, is frequently used as a nanocarrier due to its biocompatibility, immunostimulating properties, non-toxicity, biodegradability, adhesive properties, and relatively low cost of production [115, 118]. Chitosan has a high ability to inhibit biofilm growth because of its polycationic nature, which results in electrostatic interaction with the biofilm components and damages the biofilm matrix [119]. Additionally, electrostatic interaction between positively charged chitosan and negatively charged bacterial cell surfaces leads to the destruction of bacterial cell membranes and the leakage of their constituent parts, as well as the inhibition of mRNA transcription and protein synthesis through DNA binding [100].

#### **5.4 pH-responsive polymer-based nanoparticles**

The ability to be functionalized in accordance with the conditions of the microenvironment determines the polymeric NPs. As previously discussed, the rapid pH-responsive transmission of the NPs negative to positive charge increases their capacity to accumulate and penetrate biofilms inside acidic microenvironments, which decreases drug efficacy. In addition to effectively binding to the bacterial cell surfaces and enhancing photoinactivation efficiency against Gram-negative bacteria, the pH-responsive polymeric NPs carriers have a high potential to interact with the acidic biofilm microenvironment and respond to pH variation [120, 121]. The acidic pH-responsive NPs systems have been developed as bilayers with a cationic outer shell for binding with EPS components and a hydrophobic inner shell for releasing the encapsulated drug and enhancing antimicrobial and antibiofilm activity [120, 122]. For example, Horev et al. [120] conducted an in vitro and in vivo study that demonstrated farnesol-loaded pH-activated polymer NPs had a 4-fold greater ability to inhibit *S. mutans* biofilms than free farnesol; additionally, the drug was concentrated at the biofilm-EPS matrix interface, which greatly improved farnesol retention and bioavailability [120].
